1. Field of the Invention
The present invention relates to novel transglutaminase preparations derivable from the class Oomycetes, a novel transglutaminase derived from Phytophthora cactorum, CBS 618.94 or IFO 30474, a DNA construct encoding the transglutaminase enzyme, a method of producing the novel transglutaminase and the novel transglutaminase preparation, a method for producing a gel or protein gelation composition, and the use thereof.
2. Description of Related Art
Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor. Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted gamma-amides of peptide-bound glutamic acid. When the xcex5-amino group of a lysine residue in a peptide-chain serves as the acyl acceptor, the transglutaminases form intramolecular or intermolecular xcex5-(xcex3-Glu)-Lys crosslinks.
This peptide crosslinking activity is useful for a variety of industrial purposes, including gelling of proteins, reduction of antigenicity of proteins, improvement of baking quality of flour, producing paste type food materia from protein, fat and water, preparation of cheese from milk concentrate, binding of chopped meat product, improvement of taste and texture of food proteins, producing jelly, gel cosmetics etc.
A wide array of transglutaminases have been isolated and characterized from animals and plants. The animal derived TGases are Ca2+-dependent and often multi-subunit enzymes. The most widely used mammalian transglutaminase, FXIIIa, is product inhibited, difficult to obtain in high amounts and thus expensive, and therefore not useful for all applications.
A few microbial TGases have been described, including the Ca2+-independent TGases from Streptoverticillia disclosed in U.S. Pat. No. 5,156,956 and related species disclosed in U.S. Pat. No. 5,252,469.
The yields of the microbial transglutaminases in shake-flasks and fermentors are far below those seen for other industrial enzymes. Thus, better production methods, including new high-yielding producers are needed. Previously, this goal has been pursued by applying conventional recombinant DNA techniques for cloning and expression in E. coli, S. cerevisiae and S. lividans (Washizu et al.; Tahekana et al.; Takagi et al.) but without success.
Klein et al. found and partially characterized a transglutaminase from the slime mold Physarum polycephalum which is a homodimer having a total molecular weight of 77 kDa. JP 6078783 Kokai relates to the use of this transglutaminase for protein gelation. However, it is well-known that slime molds are unsuited for large-scale industrial fermentation. Further, Physarum is not a fungus; it belongs to the Myxomycetes (Entrez NIH data base, current version January 1996). Taxonomically, the only common feature of Oomycetes, Myxomycetes and Eumycota (fungi) is that they all are mitochondrial eukaryotes.
The object of the invention is to provide a novel transglutaminase, a novel transglutaminase preparation, a method for producing the transglutaminase or transglutaminase preparation in a better yield and higher purity than hitherto possible which transglutaminase can be used either alone or in combination with other enzymes for industrial purposes.
Surprisingly, it has been found that organisms belonging to the class Oomycetes produce transglutaminase and that high-level expression is achievable by growing these coenocytium forming organisms.
In particular, isolates belonging to the class Oomycetes have been shown to express transglutaminases in unprecedented high amounts, including isolates belonging to the order Peronosporales, family Pythiaceae, and the genera Pythium and Phytophthora.
Accordingly, the present invention relates to transglutaminase preparations producible by cultivation of a transglutaminase producing strain of the class Oomycetes and to novel transglutaminases derived from transglutaminase producing strains of the class Oomycetes. Preferably, the novel transglutaminase and the transglutaminase preparation of the invention are derived from or producible by transglutaminase producing strains belonging to the class Oomycetes.
Further, the present invention relates to a parent transglutaminase derived from or producible by a species selected from Phytophthora cactorum, CBS 618.94 or IFO 30474, Phytophthora cryptogea, CBS 651.94, Pythium periilum (or P. periplocum), CBS 620.94, Pythium irregulars, CBS 701.95, Pythium sp., CBS 702.95, Pythium intermedium, CBS 703.95, Pythium sp., CBS 704.95, Pythium ultimum, CBS 705.95 or a functional analogue thereof.
The present invention also relates to a method for the production of a transglutaminase preparation according to the invention by cultivating, in a suitable medium, a strain belonging to the class Oomycetes, preferably belonging to an order selected from Peronosporales, Saprolegniales, Leptomitales and Lagenidiales, more preferably belonging to a family selected from Pythiaceae, Peronosporaceae, Saprolegniaceae, Leptomitaceae, Rhiphidiaceae and Lagenidiaceae, especially belonging to a genus selected from Pythium and Phytophthora.
Further, the present inventors have now surprisingly succeeded in isolating and characterizing a DNA sequence from a strain of the oomycetes Phytophthora cactorum exhibiting transglutaminase activity, thereby making it possible to prepare a recombinant transglutaminase.
Accordingly, in yet another aspect the invention relates to a DNA construct comprising a DNA sequence encoding an enzyme exhibiting transglutaminase activity, which DNA sequence comprises
a) the DNA sequence shown in SEQ ID No. 1, and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256 or
b) an analogue of the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, which
i) is homologous with the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, or
ii) hybridizes with the same oligonucleotide probe as the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Eschefichia coli DSM 10256, or
iii) encodes a polypeptide which is homologous with the polypeptide encoded by a DNA sequence comprising the DNA sequence shown in SEQ ID No. 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256, or
iv) encodes a polypeptide which is immunologically reactive with an antibody raised against the purified transglutaminase encoded by the DNA sequence shown in SEQ ID No 1 and/or the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256.
It is believed that the DNA sequence shown in SEQ ID No. 1 is identical to the DNA sequence obtainable from the plasmid in Escherichia coli DSM 10256.
The strain Escherichia coli was deposited under the deposition number DSM 10256 on Sep. 18, 1995 at the DSMxe2x80x94Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maascheroder Weg 1b, D-38125 Braunschweig, Germany, according to the Budapest Treaty.
In another aspect, the invention relates to a method of crosslinking proteins comprising contacting a proteinaceous substrate with a transglutaminase or transglutaminase preparation of the present invention.
In yet another aspect, the invention relates to use of the transglutaminase or transglutaminase preparation of the present invention in flour, baked products, meat products, fish products, cosmetics, cheese, milk products, gelled food products and leather finishing.
In the present specification and claims, the term xe2x80x9ctransglutaminasexe2x80x9d is intended to be understood as an enzyme capable of catalyzing an acyl transfer reaction in which a gamma-carboxyamide group of a peptide-bound glutamine residue is the acyl donor.
In the present context the term xe2x80x9cderivablexe2x80x9d or xe2x80x9cderived fromxe2x80x9d is intended not only to indicate a transglutaminase produced by a strain of the organism in question, but also a transglutaminase encoded by a DNA sequence isolated from such strain and produced in a host organism transformed with said DNA sequence. Furthermore, the term is intended to indicate a transglutaminase which is encoded by a DNA sequence of synthetic and/or cDNA origin and which has the identifying characteristics of the transglutaminase in question.
The transglutaminase may be a component occurring in an enzyme system produced by a given microorganism, such an enzyme system mostly comprising several different enzyme components. In the present specification and claims, such an enzyme system comprising at least one transglutaminase component is denoted xe2x80x9ctransglutaminase preparationxe2x80x9d.
Alternatively, the transglutaminase may be a single component, i.e. a component essentially free of other enzyme components usually occurring in an enzyme system produced by a given microorganism, the single component being a recombinant component, i.e. produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host. The host is preferably a heterologous host, but the host may under certain conditions also be the homologous host. A recombinant transglutaminase may be cloned and expressed according to standard techniques conventional to the skilled person.
According to the present invention, the native or unmodified transglutaminase is of microbial origin, more specifically obtainable from a strain belonging to the class Oomycetes.
The class Oomycetes comprises the orders Peronosporales, Saprolegniales, Leptomitales and Lagenidiales.
The order Peronosporales comprises the families Pythiaceae, Peronosporaceae, Peronophytophthoraceae and Albuginaceae.
The order Saprolegniales comprises the families Saprolegniaceae, Ectrogellaceae, Thraustochytriaceae, Haliphthoraceae and Leptolegniellaceae.
The order Leptomitales comprises the families Leptomitaceae and Rhiphidiaceae.
The order Lagenidiales comprises the families Lagenidiaceae, Olpidiaceae and Sirolpidiaceae.
It is contemplated that all orders and all families taxonomically belonging to the class Oomycetes comprise transglutaminase producing strains. In this respect it should be noted that the families Peronophytophthoraceae, Albuginaceae, Ectrogellaceae, Thraustochytriaceae, Haliphthoraceae, Leptolegniellaceae, Olpidiaceae and Sirolpidiaceae are small and often highly specialised. Thus, the families Pythiaceae, Peronosporaceae, Saprolegniaceae, Leptomitaceae, Rhiphidiaceae and Lagenidiaceae should be considered as being representative of the Oomycetes.
In a preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Pythiaceae, preferably to the genus Pythium or the genus Phytophthora, more preferably to a subdivision of the genus Pythium Pringsheim (Waterhouse) or a subdivision of the genus Phytophthora deBary (Newhook, Waterhouse and Stamps). In the following, examples of members of all subdivisions (I-III) of genus Pythium, and all subdivisions (I-VI) of genus Phytophthora are given. Examples of transglutaminase producing species of the genus Pythium are
I) P. irregulars, CBS 701.95;
IIA1) P. dissotocum; 
IIA2) P. periilum (or P. pefiplocum); P. torulosum; P. aphanidennatum; preferably P. periilum (or P. periplocum), CBS 620.94;
IIB) P. ultimum, CBS 705.95;
III) P. intermedium, CBS 703.95.
Examples of transglutaminase producing species of the genus Phytophthora are
I) P. cactorum; preferably P. cactorum, CBS 618.94 and IFO 30474.
II) P. palmivora; 
III) P. porri; 
IV) P. infestans; 
V) P. megaspenna; 
VI) P. cryptogea; and P. cinnamomi; preferably P. cryptogea, CBS 651.94.
In another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Peronosporaceae, preferably to the genus Plasmopara, more preferably to the species Plasmopara halstedii. 
In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Saprolegniaceae, preferably to a genus selected from the genera Achlya, Saprolegnia and Aphanoniyces.
In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Leptomitaceae, preferably to a genus selected from the genera Apodachlya and Leptomitus.
In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Rhiphidiaceae, preferably to a genus selected from the genera Aqualinderella and Rhiphidium.
In yet another preferred embodiment, the transglutaminase preparation of the present invention is producible by a transglutaminase producing strain which taxonomically belongs to the family Lagenidiaceae, preferably to a genus selected from the genera Lagenidium and Olpidiopsis.
In a preferred aspect of the invention, it is contemplated that novel transglutaminases are obtainable by or derivable from species selected from the group of genera consisting of Pythium and Phytophthora, more preferably from the species Pythium periilum (or P. periplocum), Pythium irregulars, Pythium sp., Pythium ultimum, Pythium intermedium, Phytophthora cactorum and Phytophthora cryptogea, especially from the species Pythium periilum (or P. periplocum) deposited at Centraalbureau voor Schimmelcultures, Oosterstraat 1, NL-3742 SK Baarn, The Netherlands on Dec. 20, 1994 under the deposition number CBS 620.94; Phytophthora cactorum deposited at Centraalbureau voor Schimmelcultures under the deposition number CBS 618.94 on Dec. 20, 1994 (and redeposited on Oct. 19, 1995) and previously at the Institute for Fermentation, Osaka, under the deposition number IFO 30474; Phytophthora cryptogea deposited at Centraalbureau voor Schimmelcultures on Dec. 27, 1994 under the deposition number CBS 651.94; Pythium irregulare deposited at Centraalbureau voor Schimmelcultures on Oct. 19, 1995 under the deposition number CBS 701.95; Pythium sp. deposited at Centraalbureau voor Schimmelcultures on Oct. 19, 1995 under the deposition number CBS 702.95; Pythium internedium deposited at Centraalbureau voor Schimmelcultures on Oct. 19, 1995 under the deposition number CBS 703.95; Pythium sp. deposited at Centraalbureau voor Schimmelcultures on Oct. 19, 1995 under the deposition number CBS 704.95; Pythium ultimum deposited at Centraalbureau voor Schimmelcultures on Oct. 19, 1995 under the deposition number CBS 705.95; all depositions made under the Budapest Treaty.
The transglutaminase component may be derived either from the homologous or a heterologous host. Preferably, the component is homologous. However, a heterologous component which is immunologically reactive with an antibody raised against a highly purified transglutaminase and which is derived from a specific microorganism is also preferred.
Advantageously, a parent transglutaminase derivable from a strain of the genera Pythium and Phytophthora may be used.
In a preferred embodiment, the parent transglutaminase is selected from the group consisting of a Phytophthora cactorum, CBS 618.94/IFO 30474, transglutaminase; a Pythium pertilum (or P. periplocum), CBS 620.94, transglutaminase; a Pythium irregulars, CBS 701.95, transglutaminase; a Pythium sp., CBS 702.95, transglutaminase; a Pythium intermedium, CBS 703.95, transglutaminase; a Pythium sp., CBS 704.95, transglutaminase; a Pythium ultimum, CBS 705.95, transglutaminase and a Phytophthora cryptogea, CBS 651.94, transglutaminase; or is a functional analogue of any of said parent transglutaminases which
(i) comprises an amino acid sequence being at least 40%, preferably at least 60%, especially more than 74%, homologous with the amino acid sequence of the parent transglutaminase,
(ii) reacts with an antibody raised against the parent transglutaminase, and/or
(iii) is encoded by a DNA sequence which hybridizes with the same probe as a DNA sequence encoding the parent transglutaminase.
Property i) of the analogue is intended to indicate the degree of identity between the analogue and the parent transglutaminase indicating a derivation of the first sequence from the second. In particular, a polypeptide is considered to be homologous to the parent transglutaminase if a comparison of the respective amino acid sequences reveals an identity of greater than about 40%, such as above 45%, 50%, 55%, 60%, 65%, 70%, 74%, 80%, 85%, 90% or even 95%. Sequence comparisons can be performed via known algorithms, such as the one described by Lipman and Pearson (1985).
The additional properties ii) and iii) of the analogue of the parent transglutaminase may be determined as follows:
Property ii), i.e. the immunological cross reactivity, may be assayed using an antibody raised against or reactive with at least one epitope of the parent transglutaminase.
The antibody, which may either be monoclonal or polyclonal, may be produced by methods known in the art, e.g. as described by Hudson et al., 1989. The immunological cross-reactivity may be determined using assays known in the art, examples of which are Western Blotting or radial immunodiffusion assay, e.g. as described by Hudson et al., 1989.
The probe used in the characterization of the analogue in accordance with property iii) defined above, may suitably be prepared on the basis of the full or partial nucleotide or amino acid sequence of the parent transglutaminase. The hybridization may be carried out under any suitable conditions allowing the DNA sequences to hybridize. For instance, such conditions are hybridization under specified conditions, e.g. involving presoaking in 5xc3x97SSC and prehybridizing for 1 h at xcx9c45xc2x0 C. in a solution of 5xc3x97SSC, 5xc3x97Denhardt""s solution, 0.5% SDS, and 100 xcexcg/ml of denatured sonicated salmon sperm DNA, followed by hybridization in the same solution supplemented with 32P-dCTP-labelled probe for 12 h at xcx9c45xc2x0 C., or other methods described by e.g. Sambrook et al., 1989.
In the present context, the xe2x80x9canaloguexe2x80x9d of the DNA sequence shown in SEQ ID No. 1 is intended to indicate any DNA sequence encoding an enzyme exhibiting transglutaminase activity, which has any or all of the properties i)-iv) of claim 27. The analogous DNA sequence
a) may be isolated from another or related (e.g. the same) organism producing the enzyme with transglutaminase activity on the basis of the DNA sequence shown in SEQ ID No. 1, e.g. using the procedures described herein, and thus, e.g. be an allelic or species variant of the DNA sequence comprising the DNA sequences shown herein,
b) may be constructed on the basis of the DNA sequence shown in SEQ ID No. 1, e.g. by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the transglutaminase encoded by the DNA sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions which may give rise to a different amino acid sequence. However, in the latter case amino acid changes are preferably of a minor nature, that is conservative amino acid substitutions that do not significantly affect the folding or activity of the protein, small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain. See in general Ford et al., Protein Expression and Purification 2: 95-107, 1991. Examples of conservative substitutions are within the group of basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, valine), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, threonine, methionine).
It will be apparent to persons skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acids essential to the activity of the polypeptide encoded by the DNA construct of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244, 1081-1085, 1989). In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological (i.e. transglutaminase) activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photoaffinity labeling. See, for example, de Vos et al., Science 255: 306-312, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992.
The homology referred to in i) above or of claim 27 is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S. B. and Wunsch, C. D., Journal of Molecular Biology, 48: 443-453, 1970). Using GAP with the following settings for DNA sequence comparison: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the DNA sequence exhibits a degree of identity preferably of at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 74%, even more preferably at least 80%, especially at least 90%, with the coding region of the DNA sequence shown in SEQ ID No.1.
The hybridization referred to in ii) above or of claim 27 is intended to indicate that the analogous DNA sequence hybridizes to the same probe as the DNA sequence encoding the transglutaminase enzyme under certain specified conditions which are described in detail in the Materials and Methods section hereinafter. Normally, the analogous DNA sequence is highly homologous to the DNA sequence such as at least 70% homologous to the DNA sequence shown in SEQ ID No. 1 encoding an transglutaminase of the invention, such as at least 75%, at least 80%, at least 85%, at least 90% or even at least 95% homologous to said DNA sequence.
The homology referred to in iii) above or of claim 27 is determined as the degree of identity between the two sequences indicating a derivation of the first sequence from the second. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman, S. B. and Wunsch, C. D., Journal of Molecular Biology, 48: 443-453, 1970). Using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1, the polypeptide encoded by a homologous DNA sequence exhibits a degree of identity preferably of at least 70%, more preferably at least 75%, most preferably at least 80%, especially at least 90%, with the enzyme encoded by a DNA construct comprising the DNA sequence shown in SEQ ID No. 1.
In connection with property iv) above or of claim 27 it is intended to indicate a transglutaminase encoded by a DNA sequence isolated from strain CBS 618.94 and produced in a host organism transformed with said DNA sequence or produced by the strain CBS 618.94. The immunological reactivity may be determined by the method described in the Materials and Methods section below.
In further aspects the invention relates to an expression vector harbouring a DNA construct of the invention, a cell comprising the DNA construct or expression vector and a method of producing an enzyme exhibiting transglutaminase activity which method comprises culturing said cell under conditions permitting the production of the enzyme, and recovering the enzyme from the culture.
In a still further aspect the invention relates to an enzyme exhibiting transglutaminase activity, which enzyme
a) is encoded by a DNA construct of the invention
b) produced by the method of the invention, and/or
c) is immunologically reactive with an antibody raised against a purified transglutaminase encoded by the DNA sequence shown in SEQ ID No. 1.
The transglutaminase mentioned in c) above may be encoded by the DNA sequence isolated from the strain Phytophthora cactorum, CBS 618.94, and produced in a host organism transformed with said DNA sequence or produced by the strain CBS 618.94.
The DNA sequence of the invention encoding an enzyme exhibiting transglutaminase activity may be isolated by a general method involving
cloning, in suitable vectors, a DNA library from Phytophthora cactorum, 
transforming suitable yeast host cells with said vectors,
culturing the host cells under suitable conditions to express any enzyme of interest encoded by a clone in the DNA library,
screening for positive clones by determining any transglutaminase activity of the enzyme produced by such clones, and
isolating the enzyme encoding DNA from such clones.
The general method is further disclosed in WO 94/14953 the contents of which are hereby incorporated by reference. A more detailed description of the screening method is given in Example 5 below.
The DNA sequence coding for the enzyme may for instance be isolated by screening a cDNA library of Phytophthora cactorum, and selecting for clones expressing transglutaminase activity, or from Escherichia coli, DSM 10256. The appropriate DNA sequence may then be isolated from the clone by standard procedures, e.g. as described in Example 5.
It is expected that a DNA sequence coding for a homologous enzyme, i.e. an analogous DNA sequence, is obtainable from other microorganisms. For instance, the DNA sequence may be derived by similarly screening a cDNA library of another fungus, such as a strain of Pythium.
Alternatively, the DNA coding for a transglutaminase of the invention may, in accordance with well-known procedures, conveniently be isolated from DNA from a suitable source, such as any of the above mentioned organisms, by use of synthetic oligonucleotide probes prepared on the basis of a DNA sequence disclosed herein. For instance, a suitable oligonucleotide probe may be prepared on the basis of the nucleotide sequence shown in SEQ ID No. 1 or any suitable subsequence thereof.
The DNA sequence may subsequently be inserted into a recombinant expression vector. This may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
In the vector, the DNA sequence encoding the transglutaminase should be operably connected to a suitable promoter and terminator sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. The procedures used to ligate the DNA sequences coding for the transglutaminase, the promoter and the terminator, respectively, and to insert them into suitable vectors are well known to persons skilled in the art (cf., for instance, Sambrook et al., 1989).
The host cell which is transformed with the DNA sequence encoding the enzyme of the invention is preferably a eukaryotic cell, in particular a fungal cell such as a yeast or filamentous fungal cell. In particular, the cell may belong to a species of Aspergillus or Trichodenna, most preferably Aspergillus oryzae or Aspergillus niger. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se. The use of Aspergillus as a host microorganism is described in EP 238 023 (of Novo Nordisk A/S), the contents of which are hereby incorporated by reference. The host cell may also be a yeast cell, e.g. a strain of Saccharomyces, in particular Saccharomyces cerevisiae, Saccharomyces kluyveri or Saccharomyces uvarum, a strain of Schizosaccaromyces sp., such as Schizosaccharomyces pombe, a strain of Hansenula sp. Pichia sp., Yarrowia sp. such as Yarrowia lipolytica, or Kluyveromyces sp. such as Kluyveromyces lactis. 
In a still further aspect, the present invention relates to a method of producing an enzyme according to the invention, wherein a suitable host cell transformed with a DNA sequence encoding the enzyme is cultured under conditions permitting the production of the enzyme, and the resulting enzyme is recovered from the culture.
The medium used to culture the transformed host cells may be any conventional medium suitable for growing the host cells in question. The expressed transglutaminase may conveniently be secreted into the culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
MATERIALS AND METHODS
Deposited organism:
Escherichia coli DSM 10256 containing the plasmid comprising the full length DNA sequence, coding for the transglutaminase of the invention, in the shuttle vector pYES 2.0.
Yeast Strain:
The Saccharomyces cerevisiae strain used was W3124 (MATxcex1; ura 3-52; leu 2-3, 112; his 3-D200; pep 4-1137; prc1::HIS3; prb1:: LEU2; cir+).
Plasmids:
The Aspergillus expression vector pHD414 is a derivative of the plasmid p775 (described in EP 238 023). The construction of pHD414 is further described in WO 93/11249.
Isolation of the DNA Sequence Shown in SEQ ID No. 1:
The full length DNA sequence, comprising the cDNA sequence shown in SEQ ID No. 1 coding for the transglutaminase of the invention, can be obtained from the deposited organism Escherichia coli DSM 10256 by extraction of plasmid DNA by methods known in the art (Sambrook et al.).
Extraction of total RNA was performed with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion, and isolation of poly(A)+RNA was carried out by oligo(dT)-cellulose affinity chromatography using the procedures described in WO 94/14953.
cDNA Synthesis:
Double-stranded cDNA was synthesized from 5 xcexcg poly(A)+RNA by the RNase H method (Gubler and Hoffman, Sambrook et al.) using the hair-pin modification developed by F. S. Hagen (pers. comm.). The poly(A)+RNA (5 xcexcg in 5 xcexcl of DEPC-treated water) was heated at 70xc2x0 C. for 8 min. in a pre-siliconized, RNase-free Eppendorp tube, quenched on ice and combined in a final volume of 50 xcexcl with reverse transcriptase buffer (50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, Bethesda Research Laboratories) containing 1 mM of dATP, dGTP and dTTP and 0.5 mM 5-methyl-dCTP (Pharmacia), 40 units human placental ribonuclease inhibitor (RNasin, Promega), 1.45 xcexcg of oligo(dT)18-Not I primer (Pharmacia) and 1000 units SuperScript II RNase H reverse transcriptase (Bethesda Research Laboratories). First-strand cDNA was synthesized by incubating the reaction mixture at 45xc2x0 C. for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gelfiltrated through a MicroSpin S-400 HR (Pharmacia) spin column according to the manufacturer""s instructions.
After the gelfiltration, the hybrids were diluted in 250 xcexcl second strand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl2, 10 mM (NH4)2SO4, 0.16 mM xcex2NAD+) containing 200 xcexcM of each dNTP, 60 units E. coli DNA polymerase I (Pharmacia), 5.25 units RNase H (Promega) and 15 units E. coli DNA ligase (Boehringer Mannheim). Second strand cDNA synthesis was performed by incubating the reaction tube at 16xc2x0 C. for 2 hours and additional 15 min. at 25xc2x0 C. The reaction was stopped by addition of EDTA to a final concentration of 20 mM followed by phenol and chloroform extractions.
Mung bean Nuclease Treatment:
The double-stranded cDNA was precipitated at xe2x88x9220xc2x0 C. for 12 hours by addition of 2 vols 96% EtOH, 0.2 vol 10 M NH4Ac, recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 xcexcl Mung bean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO4, 0.35 mM DTT, 2% glycerol) containing 25 units Mung bean nuclease (Pharmacia). The single-stranded hair-pin DNA was clipped by incubating the reaction at 30xc2x0 C. for 30 min., followed by addition of 70 xcexcl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA, phenol extraction and precipitation with 2 vols of 96% EtOH and 0.1 vol 3 M NaAc, pH 5.2 on ice for 30 min.
Blunt-ending with T4 DNA Polymerase:
The double-stranded cDNAs were recovered by centrifugation and blunt-ended in 30 xcexcl T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM of each dNTP and 5 units T4 DNA polymerase (New England Biolabs) by incubating the reaction mixture at 16xc2x0 C. for 1 hour. The reaction was stopped by addition of EDTA to a final concentration of 20 mM, followed by phenol and chloroform extractions, and precipitation for 12 hours at xe2x88x9220xc2x0 C. by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.
Adaptor Ligation, Not I Digestion and Size Selection:
After the fill-in reaction the cDNAs were recovered by centrifugation, washed in 70% EtOH and dried. The cDNA pellet was resuspended in 25 xcexcl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP) containing 2.5 xcexcg non-palindromic BstXI adaptors (Invitrogen) and 30 units T4 ligase (Promega) and incubated at 16xc2x0 C. for 12 hours. The reaction was stopped by heating at 65xc2x0 C. for 20 min. and then cooling on ice for 5 min. The adapted cDNA was digested with Not I restriction enzyme by addition of 20 xcexcl water, 5 xcexcl 10xc3x97 Not I restriction enzyme buffer (New England Biolabs) and 50 units Not I (New England Biolabs), followed by incubation for 2.5 hours at 37xc2x0 C. The reaction was stopped by heating at 65xc2x0 C. for 10 min. The cDNAs were size-fractionated by gel electrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarose gel (FMC) in 1xc3x97TBE to separate unligated adaptors and small cDNAs. The cDNA was size-selected with a cut-off at 0.7 kb and rescued from the gel by use of xcex2-Agarase (New England Biolabs) according to the manufacturer""s instructions and precipitated for 12 hours at xe2x88x9220xc2x0 C. by adding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.
Construction of Libraries:
The directional, size-selected cDNA was recovered by centrifugation, washed in 70% EtOH, dried and resuspended in 30 xcexcl 10 mM Tris-Cl, pH 7.5, 1 mM EDTA. The cDNAs were desalted by gelfiltration through a MicroSpin S-300 HR (Pharmacia) spin column according to the manufacturer""s instructions. Three test ligations were carried out in 10 xcexcl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 0.5 mM ATP) containing 5 xcexcl double-stranded cDNA (reaction tubes #1 and #2), 15 units T4 ligase (Promega) and 30 ng (tube #1), 40 ng (tube #2) and 40 ng (tube #3, the vector background control) of BstXI-NotI cleaved pYES 2.0 vector. The ligation reactions were performed by incubation at 16xc2x0 C. for 12 hours, heating at 70xc2x0 C. for 20 min. and addition of 10 xcexcl water to each tube. 1 xcexcl of each ligation mixture was electroporated into 40 xcexcl electrocompetent E. coli DH1OB cells (Bethesda research Laboratories) as described (Sambrook et al.). Using the optimal conditions a library was established in E. coli consisting of pools containing 15.000-30.000 colony forming units. Each pool of transformed E. coli was spread on LB+ampicillin agar plates giving 15.000-30.000 colonies/plate after incubation at 37xc2x0 C. for 24 hours. 20 ml LB+ampicillin was added to the plate and the cells were suspended herein. The cell suspension was shaked in a 50 ml tube for 1 hour at 37xc2x0 C. Plasmid DNA was isolated from the cells according to the manufacturer""s instructions using QIAGEN plasmid kit and stored at xe2x88x9220xc2x0 C.
1 xcexcl aliquots of purified plasmid DNA (100 ng/xcexcl) from individual pools were transformed into S. cerevisiae W3124 by electroporation (Becker and Guarante) and the transformants were plated on SC agar containing 2% glucose and incubated at 30xc2x0 C.
Identification of positive colonies:
After 3-5 days of growth, the agar plates were replica plated onto a set of SC-variant agar plates. These plates were incubated for 6-8 days at 30xc2x0 C.
Round (diameter 8.2 cm) Immobilon PVDF Transfer Membranes for protein blotting (Millipore) were wetted for 1-3 seconds in 96% EtOH and rinsed in water for 1 min. The membranes were incubated for 2 hours in 2% N,N-dimethylcasein, 150 mM NaCl, 0.1 M Trisbuffer pH 7.5 and washed twice (1 min.) in 150 mM NaCl, 0.1 M Trisbuffer pH 7.5.
A casein saturated membrane was placed on each SC-variant agar plate with yeast colonies. The plate was incubated at 30xc2x0 C. over night with 1 ml 0.5 mM 5-(biotinamido)-pentylamine (Pierce), 0.1 M Trisbuffer pH 7.5, 50 mM CaCl2. After 3 washes (15 min.) in 0.1 M Na3PO4/H3PO4 buffer pH 6.5 the membrane was incubated for 1 hour at room temperature with 10 ml 0.17 xcexcg/ml peroxidase-labeled Streptavidin (Kir{acute over (k)}egaard and Perry Laboratories Inc.). After further 3 washes (15 min.) in 0.1 M Na3PO4/H3PO4 buffer pH 6.5 the membrane was incubated at room temperature with 1 ml 2 mM ABTS (Sigma), 1 mM H2O2, 0.1 M Na3PO4/H3PO4 buffer pH 6.5 until transglutaminase positive colonies were identified by a green or lilac zone.
Cells from enzyme-positive colonies were spread for single colony isolation on agar, and an enzyme-producing single colony was selected for each of the transglutaminase-producing colonies identified.
Characterization of Positive Clones:
The positive clones were obtained as single colonies, the cDNA inserts were amplified directly from the yeast colony using biotinylated polylinker primers, purified by magnetic beads (Dynabead M-280, Dynal) system and characterized individually by sequencing the 5xe2x80x2-end of each cDNA clone using the chain-termination method (Sanger et al.) and the Sequenase system (United States Biochemical).
Isolation of a cDNA Gene for Expression in Aspergillus:
A transglutaminase-producing yeast colony was inoculated into 20 ml YPD broth in a 50 ml glass test tube. The tube was shaken for 2 days at 30xc2x0 C. The cells were harvested by centrifugation for 10 min. at 3000 rpm.
DNA was isolated according to WO 94/14953 and dissolved in 50 xcexcl water. The DNA was transformed into E. coli by standard procedures. Plasmid DNA was isolated from E. coli using standard procedures, and analyzed by restriction enzyme analysis. The cDNA insert was excised using appropriate restriction enzymes and ligated into an Aspergillus expression vector.
Transformation of Aspergillus oryzae or Aspergillus niger 
Protoplasts may be prepared as described in WO 95/02043, p. 16, line 21xe2x80x94page 17, line 12.
100 xcexcl of protoplast suspension is mixed with 5-25 xcexcg of the appropriate DNA in 10 xcexcl of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5, 10 mM CaCl2). Protoplasts are mixed with p3SR2 (an A. nidulans amdS gene carrying plasmid). The mixture is left at room temperature for 25 minutes. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl2 and 10 mM Tris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85 ml of the same solution is added and carefully mixed. The mixture is left at room temperature for 25 minutes, spun at 2500 g for 15 minutes and the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one more sedimentation the protoplasts are spread on minimal plates (Cove) containing 1.0 M sucrose, pH 7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibit background growth. After incubation for 4-7 days at 37xc2x0 C. spores are picked and spread for single colonies. This procedure is repeated and spores of a single colony after the second reisolation is stored as a defined transformant.
Test of A. Oryzae Transformants
Each of the transformants were inoculated in 10 ml YPM and propagated. After 2-5 days of incubation at 37xc2x0 C., 10 ml supernatant was removed. The transglutaminase activity was identified by the 5-(biotinamido)-pentylamine plate assay described above and the Putrescine assay described in Example 1 below.
Hybridization conditions (to be used in evaluating property ii) of the DNA construct of the invention):
Suitable conditions for determining hybridization between a DNA or RNA or an oligonucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5xc3x97SSC (standard saline citrate) for 10 min. and prehybridizing of the filter in a solution of 5xc3x97SSC (Sambrook et al., 1989), 5xc3x97Denhardt""s solution (Sambrook et al., 1989), 0.5% SDS and 100 xcexcg/ml of denatured sonicated salmon sperm DNA (Sambrook et al., 1989), followed by hybridization in the same solution containing a random-primed (Feinberg and Vogelstein, 1983) 32P-dCTP labelled (specific activity greater than 1xc3x97109 cpm/xcexcg) probe for 12 h at xcx9c45xc2x0 C. The filter is then washed two times for 30 minutes in 2xc3x97SSC, 0.5% SDS at a temperature preferably not higher than 45xc2x0 C., more preferably not higher than 50xc2x0 C., even more preferably not higher than 55xc2x0 C., even more preferably not higher than 60xc2x0 C., most preferably not higher than 65xc2x0 C., especially not higher than 70xc2x0 C., more preferably not higher than 75xc2x0 C.
A suitable DNA or RNA or an oligonucleotide probe to be used in the hybridization may be prepared on the basis of the DNA sequence shown in SEQ ID No. 1, or on basis of the deduced amino acid sequence shown in SEQ ID No.2.
Immunological Cross-reactivity:
Antibodies to be used in determining immunological cross-reactivity may be prepared by use of a purified transglutaminase. More specifically, antiserum against the transglutaminase of the invention may be raised by immunizing rabbits (or other rodents) according to the procedure described by N. Axelsen et al., Chapter 23, or A. Johnstone and R. Thorpe. Purified immunoglobulins may be obtained from the antisera, for example by salt precipitation ((NH4)2 SO4), followed by dialysis and ion exchange chromatography, e.g. on DEAE-Sephadex. Immunochemical characterization of proteins may be done either by Outcherlony double-diffusion analysis (O. Ouchterlony), by crossed immunoelectrophoresis (N. Axelsen et al., Chapters 3 and 4), or by rocket immunoelectrophoresis (N. Axelsen et al., Chapter 2).
Media
YPD: 10 g yeast extract, 20 g peptone, H2O to 900 ml. Autoclaved, 100 ml 20% glucose (sterile filtered) added.
YPM: 10 g yeast extract, 20 g peptone, H2O to 900 ml. Autoclaved, 100 ml 20% maltodextrin (sterile filtered) added.
10xc3x97Basal salt: 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH, H2O ad 1000 ml, sterile filtered.
SC-URA: 100 ml 10xc3x97Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml 1% tryptophan, H2O ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100 ml 20% glucose or 20% galactose added.
SC-agar: SC-URA, 20 g/l agar added.
SC-variant agar: 20 g agar, 20 ml 10xc3x97Basal salt, H2O ad 900 ml, autoclaved, 10 ml 1% tryptophan, 3.6 ml 5% threonine and 100 ml 20% galactose added.
Although the useful transglutaminase preparation or the recombinant transglutaminase may be added as such it is preferred that it is formulated into a suitable composition. The transglutaminase to be used industrially may be in any form suited for the use in question, e.g. in the form of a dry powder or granulate, in particular a non-dusting granulate, a liquid, in particular a stabilized liquid, or a protected enzyme. Granulates may be produced, e.g. as disclosed in U.S. Pat. No. 4,106,991 and U.S. Pat. No. 4,661,452, and may optionally be coated by methods known in the art. Liquid enzyme preparations may, for instance, be stabilized by adding nutritionally acceptable stabilizers such as a sugar, a sugar alcohol or another polyol, lactic acid or another organic acid according to established methods. Protected enzymes may be prepared according to the method disclosed in EP 238,216. The enzyme preparation of the invention may also comprise a preservative.
Normally, for inclusion in flour, baking or baked products, meat products, cheese and other milk products, fish products, cosmestics, various gelled food, it may be advantageous that the enzyme preparation is in the form of a dry product, e.g. a non-dusting granulate, whereas for inclusion together with a liquid it is advantageously in a liquid form.
The recombinant transglutaminase and the transglutaminase preparations of the present invention may also be used in baking for improving the development, elasticity and/or stability of dough and/or the volume, crumb structure and/or anti-staling properties of the baked product. Although the transglutaminase may be used for the preparation of dough or baked products prepared from any type of flour or meal (e.g. based on rye, barley, oat or maize) the present transglutaminases have been found to be particularly useful in the preparation of dough or baked products made from wheat or comprising substantial amounts of wheat. The baked products produced with a tranglutaminase of the invention includes bread, rolls, baguettes and the like. For baking purposes the transglutaminase of the invention may be used as the only or major enzymatic activity, or may be used in combination with other enzymes such as a lipase, an amylase, an oxidase (e.g. glucose oxidaase, peroxidase), a laccase and/or a protease.
Preferably, the transglutaminase of the invention, especially the recombinant transglutaminase, is used in flour, dough, baked products, meat products, cheese and other milk products, fish products, cosmetics, and various gelled food products in an amount of between 0.01 and 100 mg per kg, more preferably of between 0.1 and 50 mg per kg, most preferably between 0.5 and 30 mg per kg, especially between 1 and 10 mg per kg.
Further, it is contemplated that the recombinant transglutaminase and the transglutaminase preparations of the present invention also can exhibit glutaminase activity, i.e. are capable of glutamine-specific deamidation. Accordingly, a protein substrate essentially free of lysine or at least with a very low content of lysine may be subjected to deamidation by applying the transglutaminase of the invention, such as protein being e.g. gluten or a gluten hydrolysate. In another aspect of the invention, the transglutaminases of the invention can be useful for treatment of food products containing gluten, e.g. for improvement of the palability or other properties of bread and other baked food products, or for reducing the allergenicity of food products containing gluten or gluten hydrolysates.